Peripheral nerve extract promotes long-term survival and neurite outgrowth in cultured spinal cord neurons

The hypothesis that peripheral, skeletal muscle tissue contains a trophic factor supporting central neurons has recently been investigated in vitro by supplementing the culture medium of spinal cord neurons with muscle extracts and fractions of extract. We extended these studies asking whether or not a trophic factor is present in peripheral nerves, the connecting link between muscle and central neurons via which factors may be translocated from muscle to neurons by the retrograde transport system. Lumbar, 8-day-old chick spinal cords were dissociated into single cells and then cultured in the presence of peripheral nerve extract. Cytosine arabinoside was added to inhibit proliferation of nonneuronal cells. In the presence of nerve extract, spinal cord neurons survived for more than a month, extended numerous neurites, and showed activity of choline acetyltransferase. In the absence of extract, neurons attached and survived for a few days but then died subsequently in less than 10 days. Neurite outgrowth did not occur in the absence of extract. Withdrawal of extract from the medium of established neuronal cultures caused progressive loss of both cells and neurites. Other tissues also contained neuron supporting activity but less than that found in nerve extract. These studies indicate that peripheral nerves contain relatively high levels of spinal cord neuron-directed trophic activity, suggesting translocation of neurotrophic factor from muscle to central target neurons. The neurotrophic factor has long-term (weeks) effects, whereas short-term (days) survival is factor independent.     

L-arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells

L-Arginine is required for expression of the activated macrophage cytotoxic effector mechanism that causes inhibition of mitochondrial respiration, aconitase activity, and DNA synthesis in tumor target cells. This effector mechanism is active in the presence of L-arginine even when the cocultivation medium lacks all other amino acids and serum. Cytotoxic activated macrophage-induced inhibition of mitochondrial respiration in target cells is proportional to the concentration of L-arginine in the medium. L-Arginine must be present during the cocultivation period. Pretreatment of cytotoxic activated macrophages with L-arginine or posttreatment of the target cells after cocultivation is not effective. D-Arginine does not substitute for L-arginine and at high concentrations is a competitive inhibitor of the L-arginine-dependent effector mechanism. Other analogues that could not replace L-arginine include agmatine, argininic acid, arginine hydroxamate, and tosyl-L-arginine methyl ester. L-homoarginine, however, can effectively substitute for L-arginine. NG-monomethyl-L-arginine is a potent competitive inhibitor of this effector mechanism. High concentrations of lipopolysaccharide do not reverse inhibition of the L-arginine-dependent effector mechanism by NG-monomethyl-L-arginine. However, inhibition of the effector mechanism by NG-monomethyl-L-arginine can be overridden by increasing the concentration of L-arginine in the culture medium. We compared NGNG-dimethyl-L-arginine and NGN1G-dimethyl-L-arginine with NG-monomethyl-L-arginine as inhibitors of the L-arginine-dependent effector mechanism. The results show that the inhibitory effect of these guanidino methylated derivatives of L-arginine is highly determined by structure. Guanidine is a weak competitive inhibitor of the L-arginine-dependent effector mechanism. The requirement for L-arginine does not appear to be for protein synthesis, creatine biosynthesis, polyamine biosynthesis, or ADP ribosylation reactions. Bacterial lipopolysaccharide is effective as a second signal only when the cocultivation medium contains L-arginine, and this strict L-arginine dependency is not overridden by increasing the concentration of lipopolysaccharide. Bovine liver arginase, by competing for L-arginine in the cocultivation medium, inhibits the L-arginine-dependent activated macrophage cytotoxic effector mechanism.     

Inhibition by 2-deoxy-D-ribose of DNA synthesis and growth in Raji cells

When Raji cells were cultured for 3 days in serum-free medium, addition of 2-deoxy-D-ribose at the start of culture inhibited incorporation of [3H]thymidine and cell division. At deoxyribose concentrations between 1 and 5 mM, viability was 80% or greater after 3 days of culture even though 5 mM deoxyribose inhibited thymidine incorporation 95-99%. Inhibition by deoxyribose could be completely reversed if the culture medium was replaced with fresh medium up to 8 hr after the start of culture. The inhibition was specific for deoxyribose since other monosaccharides had no effect. Inhibition of DNA synthesis did not appear to be due to depletion of essential nutrients in the medium since the percentage inhibition of thymidine incorporation by cells cultured either in suboptimal serum-free media or in media supplemented with 0.025-5% human AB serum was similar. When DNA repair synthesis was measured as hydroxyurea-resistant thymidine incorporation, addition of deoxyribose to Raji cultures caused increased thymidine incorporation. These results, together with data from others, suggest that deoxyribose damages DNA.     

A clonogenic survival assay of neural stem cells in rat spinal cord after exposure to ionizing radiation

Neural stem cells play an important role in neurogenesis of the adult central nervous system (CNS). Inhibition of neurogenesis has been suggested to be an underlying mechanism of radiation-induced CNS damage. Here we developed an in vivo/ in vitro clonogenic assay to characterize the survival of neural stem cells after exposure to ionizing radiation. Cells were isolated from the rat cervical spinal cord and plated as single cell suspensions in defined medium containing epidermal growth factor and basic fibroblast growth factor. The survival of the proliferating cells was determined by their ability to form neurosphere colonies. The number and size of neurospheres were analyzed quantitatively at day 10, 12, 14 and 16 after plating. Plating cells from 5, 10 and 15 mm of the cervical spinal cord resulted in a linear increase in the number of neurospheres from day 10-16. Compared to the nonirradiated spinal cord, there was a significant decrease in the number and size of neurosphere colonies cultured from a 10-mm length of the rat spinal cord after a single dose of 5 Gy. When dissociated neurospheres derived from a spinal cord that had been irradiated with 5 Gy were allowed to differentiate, the percentages of neurons, oligodendrocytes and astrocytes as determined by immunocytohistochemistry were not altered compared to those from the nonirradiated spinal cord. Secondary neurospheres could be obtained from cells dissociated from primary neurospheres that had been cultured from the irradiated spinal cord. In conclusion, exposure to ionizing radiation reduces the clonogenic survival of neural stem cells cultured from the rat spinal cord. However, neural stem cells retain their pluripotent and self-renewing properties after irradiation. A neurosphere-based assay may provide a quantitative measure of the clonogenic survival of neural stem cells in the adult CNS after irradiation.     

Neurite outgrowth and proliferation of non-neuronal cells on cryostat sections of adult muscle

Rat spinal cord cells were cultured on cryostat sections of innervated and denervated muscle. Neurite outgrowth was greater on sections of denervated muscle, which therefore appeared to act on in vivo nerve regeneration. It seems that muscle sections were able to release into the culture medium factors that increase proliferation of fibroblasts. The muscle therefore appeared able to modulate its interaction with its environment by acting on different types of cells.     

AN ANALYSIS OF THE EFFECT OF "CONDITIONED MEDIUM" UPON THE CELL CULTURE AT LOW DENSITY

A favorable effect of “conditioned medium” upon outgrowth of the cell culture with low density in vitro was analysed with the cells of chicken embryos. For preparing “conditioned medium”, cultures with a large number of cells were made with muscle, kidney, lung, liver and skin, while the biological activity of the medium was assayed by using the culture of a small number of the lung secondary cells. A use of “conditioned medium” was found to be necessary for encouraging the outgrowth of the cultured cells below a critical inoculum size. Of the various types of the media tested, the medium conditioned with muscle was most effective. “Conditioned medium” contained at least two different active factors, the first to enhance the plating efficiency of the inoculated cells to the surface of the culture dish, and the second to promote further outgrowth of the plated cells. “Conditioned medium” taken out of the mass culture at its exponentially growing phase had only the second factor, while that taken out of that at its stationary phase contained both factors. An activity of the first factor was not detected, when the mass culture was kept in such condition that the collagen synthesis was inhibited. The factor for enhancing the plating efficiency was eliminated from “conditioned medium” by preincubating the cells, before assaying the effect of the medium.     

Neurite outgrowth and proliferation of non-neuronal cells on cryostat sections of adult muscle

Rat spinal cord cells were cultured on cryostat sections of innervated and denervated muscle. Neurite outgrowth was greater on sections of denervated muscle, which therefore appeared to act on in vivo nerve regeneration. It seems that muscle sections were able to release into the culture medium factors that increase proliferation of fibroblasts. The muscle therefore appeared able to modulate its interaction with its environment by acting on different types of cells.     

Dorsal spinal cord inhibits oligodendrocyte development

Oligodendrocytes are the myelinating cells of the mammalian central nervous system. In the mouse spinal cord, oligodendrocytes are generated from strictly restricted regions of the ventral ventricular zone. To investigate how they originate from these specific regions, we used an explant culture system of the E12 mouse cervical spinal cord and hindbrain. In this culture system O4(+) cells were first detected along the ventral midline of the explant and were subsequently expanded to the dorsal region similar to in vivo. When we cultured the ventral and dorsal spinal cords separately, a robust increase in the number of O4(+) cells was observed in the ventral fragment. The number of both progenitor cells and mature cells also increased in the ventral fragment. This phenomenon suggests the presence of inhibitory factor for oligodendrocyte development from dorsal spinal cord. BMP4, a strong candidate for this factor that is secreted from the dorsal spinal cord, did not affect oligodendrocyte development. Previous studies demonstrated that signals from the notochord and ventral spinal cord, such as sonic hedgehog and neuregulin, promote the ventral region-specific development of oligodendrocytes. Our present study demonstrates that the dorsal spinal cord negatively regulates oligodendrocyte development.     

Specific inhibition by prostaglandin D2 and its metabolites of lysozyme synthesis in mouse macrophage-like cell line, Mm-1

The cultured mouse macrophage-like cell line Mm-1 synthesizes and secretes lysozyme continuously like normal macrophages. Culture of the cells in the presence of prostaglandin D2 for 3 days strongly inhibited their production of lysozyme activity. Prostaglandin D2 caused dose-dependent inhibition of the activity: 1 X 10(-6) M prostaglandin D2 caused about 50% inhibition. Inhibition by prostaglandin D2 was not related to cytotoxicity and was reversible. The rate of synthesis of lysozyme protein was measured by culturing Mm-1 cells with radioactive amino acids and then immunoprecipitating the protein. At the concentrations used, prostaglandin D2 inhibited the synthesis of lysozyme dose-dependently, but did not suppress the synthesis of total protein. Of the various types of prostaglandin, only prostaglandin D2 inhibited the production of lysozyme in Mm-1 cells. Moreover, prostaglandin D2 did not inhibit the production of other lysosomal enzymes, such as acid proteinase, acid phosphatase and beta-glucuronidase, and did not affect Fc receptors on the cell surface, adherence of cells to the culture dish or the cell morphology. These results indicate that prostaglandin D2 specifically inhibits the synthesis of lysozyme in Mm-1 cells. When Mm-1 cells were cultured for 3 days in the presence of the ethyl acetate extract from the culture medium in which Mm-1 cells had been cultured with prostaglandin D2 for 3 days, the production of lysozyme activity of Mm-1 cells was also markedly inhibited by the extract. After the incubation of prostaglandin D2 for 3 days with Mm-1 cells, less than 10% of the initial prostaglandin D2 remained and two major metabolites appeared. These results suggest that the metabolites of prostaglandin D2 were involved in the inhibitory action of prostaglandin D2 in Mm-1 cells.     

Different proliferative potential of rat and pig hepatocytes in pure primary culture and coculture.

In the present study we have compared the growth potential of hepatocytes from rats and pigs and the influence of cocultivation between these hepatocytes and the rat liver epitheloid cell line RL-ET-14. Proliferation, i.e., DNA synthesis, was detected by autoradiography after exposure to [3H]thymidine. Rat hepatocytes cultured at low cell density showed a very low basal growth and responded to epidermal growth factor (EGF) and insulin by a considerable increase in DNA synthesis after 48 h leading to a labeling index (LI) of 33%. Cocultivation with RL-ET-14 cells almost completely blocked the basal as well as the growth factor stimulated proliferation of the rat hepatocytes. In contrast, pig hepatocytes cultured alone showed a much greater growth potential (basal: LI 11%; insulin/EGF:LI 67%) than rat hepatocytes and were further stimulated by cocultivation (basal: LI 39%; insulin/EGF: LI 89%). Density-dependent inhibition of cell growth was less pronounced with pig hepatocytes. Even after reaching confluency, they showed further strong proliferation in pure as well as in cocultures whereas the LI of the rapidly growing clone RL-ET-14 decreased to 40%. Use of conditioned medium from RL-ET-14 cells did not mimic the growth inhibition of rat hepatocytes in coculture indicating that no soluble growth inhibitors produced by the epitheloid cells are responsible for this effect. In particular, the differences between rat and pig hepatocytes in coculture are not simply due to production of TGF-beta by the epitheloid cells since the hepatocytes from both species were inhibited by TGF-beta to a similar extent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of alloantibodies on lymphocyte proliferation in vitro

Inhibition of MLC and antigen-induced proliferation by alloantibodies was studied. A serum, obtained from a multipara 3 yr after her sixth pregnancy, inhibited the capacity of the immunizer's lymphocytes to stimulate and to respond in unilateral MLC. This alloantiserum was shown to inhibit antigen-induced proliferation as well as MLC of lymphocytes from individuals unrelated to the immunizer. The inhibitory titer was much higher than the upper limit of complement dependent cytotoxicity. The inhibitory factor was found in the 7S fraction of the alloantiserum; it could be fully absorbed on leukocytes but only partially on platelets, whereas both eluates from platelets and leukocytes were inhibitory. Inhibition was still observed when the alloantiserum was added up to 3 days after the beginning of the culture, but in some instances, a potentiating affect was noticed. Lymphocytes coated with alloantibodies and then cultured with antigen did not proliferate, but replacement of the culture medium after 1 or 2 days of incubation, reversed this inhibition. In contrast, inhibition of antigen-induced transformation by heterologous antibodies was not reversible. Reversibility of the blocking effect was associated with a high rate of shedding of antibodies bound to membrane-associated structures.     

Spontaneous immortalisation of Schwann cells in culture: short-term cultured Schwann cells secrete growth inhibitory activity.

In the developing peripheral nerve, Schwann cells proliferate rapidly and then become quiescent, an essential step in control of Schwann cell differentiation. Cell proliferation is controlled by growth factors that can exert positive or inhibitory influences on DNA synthesis. It has been well established that neonatal Schwann cells divide very slowly in culture when separated from neurons but here we show that when culture was continued for several months some cells began to proliferate rapidly and non-clonal lines of immortalised Schwann cells were established which could be passaged for over two years. These cells had a similar molecular phenotype to short-term cultured Schwann cells, except that they expressed intracellular and cell surface fibronectin. The difference in proliferation rates between short- and long-term cultured Schwann cells appeared to be due in part to the secretion by short-term cultured Schwann cells of growth inhibitory activity since DNA synthesis of long-term, immortalised Schwann cells was inhibited by conditioned medium from short-term cultures. This conditioned medium also inhibited DNA synthesis in short-term Schwann cells stimulated to divide by glial growth factor or elevation of intracellular cAMP. The growth inhibitory activity was not detected in the medium of long-term immortalised Schwann cells, epineurial fibroblasts, a Schwannoma (33B), astrocytes or a fibroblast-like cell-line (3T3) and it did not inhibit serum-induced DNA synthesis in epineurial fibroblasts, 33B cells or 3T3 cells. The activity was apparently distinct from transforming growth factor-beta, activin, IL6, epidermal growth factor, atrial natriuretic peptide and gamma-interferon and was heat and acid stable, resistant to collagenase and destroyed by trypsin treatment. We raise the possibility that loss of an inhibitory autocrine loop may contribute to the rapid proliferation of long-term cultured Schwann cells and that an autocrine growth inhibitor may have a role in the cessation of Schwann cell division that precedes differentiation in peripheral nerve development.     

Neural stem/progenitor cells from the adult human spinal cord are multipotent and self-renewing and differentiate after transplantation

Neural stem/progenitor cell (NSPC) transplantation is a promising therapy for spinal cord injury (SCI). However, little is known about NSPC from the adult human spinal cord as a donor source. We demonstrate for the first time that multipotent and self-renewing NSPC can be cultured, passaged and transplanted from the adult human spinal cord of organ transplant donors. Adult human spinal cord NSPC require an adherent substrate for selection and expansion in EGF (epidermal growth factor) and FGF2 (fibroblast growth factor) enriched medium. NSPC as an adherent monolayer can be passaged for at least 9 months and form neurospheres when plated in suspension culture. In EGF/FGF2 culture, NSPC proliferate and primarily express nestin and Sox2, and low levels of markers for differentiating cells. Leukemia inhibitory factor (LIF) promotes NSPC proliferation and significantly enhances GFAP expression in hypoxia. In differentiating conditions in the presence of serum, these NSPC show multipotentiality, expressing markers of neurons, astrocytes, and oligodendrocytes. Dibutyryl cyclic AMP (dbcAMP) significantly enhances neuronal differentiation. We transplanted the multipotent NSPC into SCI rats and show that the xenografts survive, are post-mitotic, and retain the capacity to differentiate into neurons and glia.Together, these findings reveal that multipotent self-renewing NSPC cultured and passaged from adult human spinal cords of organ transplant donors, respond to exogenous factors that promote selective differentiation, and survive and differentiate after transplantation into the injured spinal cord.     

Neural agrin controls maturation of the excitation-contraction coupling mechanism in human myotubes developing in vitro

The aim of this study was to elucidate the mechanisms responsible for the effects of innervation on the maturation of excitation-contraction coupling apparatus in human skeletal muscle. For this purpose, we compared the establishment of the excitation-contraction coupling mechanism in myotubes differentiated in four different experimental paradigms: 1) aneurally cultured, 2) cocultured with fetal rat spinal cord explants, 3) aneurally cultured in medium conditioned by cocultures, and 4) aneurally cultured in medium supplemented with purified recombinant chick neural agrin. Ca(2+) imaging indicated that coculturing human muscle cells with rat spinal cord explants increased the fraction of cells showing a functional excitation-contraction coupling mechanism. The effect of spinal cord explants was mimicked by treatment with medium conditioned by cocultures or by addition of 1 nM of recombinant neural agrin to the medium. The treatment with neural agrin increased the number of human muscle cells in which functional ryanodine receptors (RyRs) and dihydropyridine-sensitive L-type Ca(2+) channels were detectable. Our data are consistent with the hypothesis that agrin, released from neurons, controls the maturation of the excitation-contraction coupling mechanism and that this effect is due to modulation of both RyRs and L-type Ca(2+) channels. Thus, a novel role for neural agrin in skeletal muscle maturation is proposed.     

Components of the translational machinery are associated with juvenile glycine receptors and are redistributed to the cytoskeleton upon aging and synaptic activity

The translation eukaryotic elongation factor 1alpha (eEF1A) is a monomeric GTPase involved in protein synthesis. In addition, this protein is thought to participate in other cellular functions such as actin bundling, cell cycle regulation, and apoptosis. Here we show that eEF1A is associated with the alpha2 subunit of the inhibitory glycine receptor in pulldown experiments with rat brain extracts. Moreover, additional proteins involved in translation like ribosomal S6 protein and p70 ribosomal S6 protein kinase as well as ERK1/2 and calcineurin were identified in the same pulldown approaches. Glycine receptor activation in spinal cord neurons cultured for 1 week resulted in an increased phosphorylation of ribosomal S6 protein. Immunocytochemistry showed that eEF1A and ribosomal S6 protein are localized in the soma, dendrites, and at synapses of cultured hippocampal and spinal cord neurons. Consistent with our biochemical data, immunoreactivities of both proteins were partially overlapping with glycine receptor immunoreactivity in cultured spinal cord and hippocampal neurons. After 5 weeks in culture, eEF1A immunoreactivity was redistributed to the cytoskeleton in about 45% of neurons. Interestingly, the degree of redistribution could be increased at earlier stages of in vitro differentiation by inhibition of either the ERK1/2 pathway or glycine receptors and simultaneous N-methyl-D-aspartate receptor activation. Our findings suggest a functional coupling of eEF1A with both inhibitory and excitatory receptors, possibly involving the ERK-signaling pathway.     

Differential effects of p-nitrophenyl-D-xylosides on mouse blastocysts and uterine epithelial cells

A number of studies have implicated glycoconjugates in cell recognition events associated with implantation of mammalian blastocysts into the uterus. We have found that p-nitrophenyl-D-xylosides inhibit mouse embryo attachment and outgrowth on monolayers of uterine epithelial cells when cocultured in vitro. Inhibition of attachment and trophoblast formation by alpha- and beta-xylosides was observed in embryos cultured on tissue culture plastic in serum containing medium or on monolayers of epithelial cells. The biochemical basis for this inhibition has been investigated. Consistent with their accepted mode of action, beta- but not alpha-D-xylosides greatly stimulated glycosaminoglycan chain production by uterine epithelial cells and likewise reduced proteoglycan assembly. In contrast, both alpha- and beta-anomers selectively inhibited embryo attachment and outgrowth without stimulating glycosaminoglycan chain production by embryos. The inhibitory effect of the xylosides on embryos was reversible and did not require concentrations that reduced the rate of protein synthesis. Both alpha- and beta-D-xylosides inhibited the synthesis of proteoglycans including heparan sulfate as well as certain other glycoconjugates by embryos. Collectively, these data indicate that proper assembly of glycoconjugates, including proteoglycans, is required for implantation-related processes, although the inhibition of embryo outgrowth by xylosides may be by an as yet uncharacterized mechanism.     

The role of cyclic GMP in cells with the properties of smooth muscle cultured from the rat myometrium

Cells growing in culture with previously described properties of rat uterine smooth muscle accumulated 45Ca2+ from the medium. Ca2+ uptake by these cells was stimulated by the addition to the medium of 8-bromo-cGMP but not by 8-bromo-cAMP. Ca2+ uptake was also stimulated by carbachol and by the nitro-vasodilator nitroprusside. Although cholinergic agonists have been shown previously to stimulate contraction but not cGMP synthesis in the rat myometrium, both carbachol and nitroprusside stimulated cGMP production by the cultured cells. These results suggested the cells had cholinergic receptor-mediated functions that reflected some neurotransmitter-sensitive properties of uterine smooth muscle in situ. When determined by a specific radioligand binding assay, subcellular fractions of the cultured cells bound muscarinic cholinergic agonists and antagonists with affinities expected of the muscarinic receptor. The cells were also sensitive to the beta-adrenergic catecholamine agonist isoproterenol, which stimulated cAMP production but not Ca2+ uptake. Carbachol failed to inhibit isoproterenol-dependent cAMP production, which is an important property of the cholinergic receptor in uterine smooth muscle in situ. These results suggest some but not all acetylcholine-sensitive properties of uterine smooth muscle may be retained in cell culture.     

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing¹. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas². To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases³, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.     

Quantitative inhibitory influence of porcine cumulus cells upon the maturation of pig and cattle oocytes in vitro

Porcine cumulus oocyte complexes (COCs) were cultured together in 10-microliters droplets of culture medium. When 10 COCs were cultured for 24 h, germinal vesicle breakdown (GVBD) occurred in 81% of them. When more COCs (20 or 40) were put into the same volume of medium the frequency of GVBD gradually decreased. This inhibition was not observed in denuded oocytes. The process of GVBD was adversely influenced when 10 COCs were cultured in cumulus-preconditioned medium. It is concluded that porcine cumulus cells produced a factor inhibiting GVBD. After removing the inhibitory block and extensive washing, GVBD of arrested oocytes was significantly accelerated. The addition of LH or heparin only partially overcame the inhibitory action. This factor produced by porcine cumulus cells negatively influenced maturation of bovine oocytes; however, a similar effect was not demonstrated in the mouse. Our results suggest that a high concentration of porcine cumulus cells exerts a quantitative inhibitory effect upon GVBD of porcine and cattle oocytes cultured in vitro.